Serotonin (5HT) is an important neuromodulator, and the serotonin transporter (SERT) plays a central role in controlling 5HT signaling and availability. Genetic variations in SERT are associated with several neuropsychiatric disorders including depression, anxiety, obsessive compulsive disorder, and autism. Moreover, SERT is a clinically relevant target for antidepressants including selective serotonin reuptake inhibitors (SSRI's), although the precise therapeutic mechanisms of these drugs remain unclear. SERT is also found in the periphery, including prominent expression in adrenal chromaffin cells where its role is poorly understood. Chromaffin cells are an important neuroendocrine component of the sympathetic nervous system that release a cocktail of catecholamines, peptides, and others transmitters to ensure coordinated physiological responses, for example during the fight-or-flight response to acute stress. They also serve as a versatile neurosecretory model that enables detailed insight into the cellular and molecular mechanisms of exocytosis. Although 5HT is typically thought to act through cell surface receptors, recently it has been proposed to act as an intracellular signaling molecule in smooth muscle, platelets, and pancreatic beta-cells. This novel mechanism involves covalent binding of 5HT to cytosolic proteins by transglutaminase-2 (dubbed serotonylation). Our preliminary data show that chromaffin cells accumulate 5HT through uptake by SERT. Our data using pharmacology (SSRIs) and transgenic mice (knock-out and knock-in models of SERT) also support a role for SERT in the regulation of chromaffin cell exocytosis. We postulate that 5HT acts at as an intracellular messenger following acute uptake by SERT to modulate neuroendocrine exocytosis. We will combine patch-clamp, carbon fiber amperometry, calcium imaging, and photolysis of caged intracellular messengers with chromaffin cells isolated from wild type mice, SERT knockout mice, and a novel knock-in mouse with a point mutation that renders SERT insensitive to antidepressants and cocaine. We will also use electron microscopy (EM) to assess the number, size, and distribution of large dense core vesicles. Biochemical approaches will determine if activity dependent serotonylation occurs. These investigations have the potential to significantly change our understanding of the mechanisms that control neurosecretion and the mechanisms of antidepressants and other drugs that target SERT. This will provide the foundation for future expanded applications to investigate a new paradigm for 5HT / SERT mediated signal transduction.